Beta-adrenergic agonists are strongly implicated in myocardial hypertrophy in vivo, and beta-adrenergic and cyclic AMP (cAMP)-dependent mechanisms are a frequent target of drugs used in ischemic cardiovascular disease and heart failure. However, the mechanisms transducing beta-adrenoceptor- mediated myocardial cell growth and gene expression are poorly understood. With few exceptions, beta-adrenergic effects in the heart have been thought to be mediated via modulation of cAMP. We have recently identified a novel pathway for beta-adrenergic gene regulation in cardiac myocytes that appears to be coupled to the storage or flux of calcium from physically or biochemically defined compartments and is independent of cAMP and cAMP-dependent protein kinase. The skeletal alpha-actin (sACT) gene, encoding a developmentally regulated actin-actin isoform, is selectively regulated by this pathway during beta-adrenoceptor-mediated hypertrophy. In order to identify essential components of this signal transduction mechanism, we propose to identify pharmacologic mechanisms for beta-adrenergic regulation of the sACT gene and to examine interactions between the sACT promoter and DNA-binding transcriptional activating factors that may be required for beta-adrenergic induction. Experiments described in part (1) will evaluate the role of beta- adrenergic, protein kinase A-independent, and calcium-dependent mechanisms in transcriptional and posttranscriptional regulation of the sACT gene, employing nuclear run-on and messenger RNA half-life assays. Because we have determined that transcriptional activating factors Fos and Jun (AP-1) positively regulates sACT in cardiac myocytes, we will determine the effects of protein kinase A-dependent, and calcium-dependent mechanisms on expression of c-fos, c-jun, and related genes as well as on AP-1 binding activity and immunoreactivity. Pl9 teratocarcinoma cells will be stably transfected with an sACT/lacZ chimeric gene containing full-length promoter sequences, most coding sequences and 3' flanking sequences in order to create a cell line in which the signal-mediated regulation of this gene can be studied directly. In part (2), beta-adrenergic and AP- 1- responsive bases in the proximal sACT promoter will be determined by a combination of in situ mutagenesis, gel mobility retardation assay, and methylation interference. Cardiac myocyte nuclear proteins will be evaluated for interaction with the sACT promoter, and a potential interaction between serum response factor and AP-1 on this promoter will be assessed. Finally, lambda-gt11 cardiac myocyte cDNA expression libraries will be constructed and screened with relevant oligonucleotide sequences from the sACT promoter.